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Abstract:

The present invention relates to computer science, molecular biology and
microfluidics technique which provides a DNA molecular computer based on
microfluidic chip. The objective is to provide a DNA molecular computer
which uses the microfluidic chip as a operation platform, mainly
including: using DNA molecules as operation media, using the microfluidic
chip as the operation platform of a DNA molecular operator; using DNA
molecules as storage media, using the microfluidic chip as the operation
platform of a DNA molecular storage; using a electronic computer and a
detector as the kernel of a controller; said microfluidic chip includes a
DNA molecular computation region and a DNA molecular storage region. The
microfluidic chip consists of digestion, ligation, PCR amplification and
chip electrophoresis unit connected by microchannels in turn, and carries
out the liquid control through micropumps and microvalves.

Claims:

1. A microfluidic chip-based DNA computer mainly comprising: using a DNA
molecule as an operation media, using the microfluidic chip as the
operation platform of a DNA molecular computation unit; using DNA
molecule as a storage media, using the microfluidic chip as the operation
platform of a DNA molecular storage; using an electronic computer and a
detector as the kernel of a controller; mentioned microfluidic chip
includes a DNA molecular computation area and a DNA molecular storage
area. The microfluidic chip is comprised of operation units of enzyme
cleavage, enzyme ligation, PCR and chip electrophoresis, which are
connected by microchannels in sequence, and carry out the liquid control
through a series of micropump and a microvalve. The controller is
connected to the electrodes of the microfluidic chips of the DNA
molecular computation unit and the DNA molecular storage unit
respectively.

2. The of claim 1 wherein mentioned DNA molecule as the operation media
completes the DNA molecular operation on the microfluidic chip of
above-mentioned DNA molecular computation unit according to the
instructions issued from above-mentioned controller.

3. The microfluidic chip-based DNA computer of claim 2 wherein: the input
part of above-mentioned DNA molecular computation unit corresponds to the
DNA computation molecule with specific sequence and the DNA transfer
molecule with specific sequence, while the output part corresponds to a
DNA output molecule that represents computation results obtained through
biochemical processes of enzyme cleavage, enzyme ligation and so on.

5. The microfluidic chip-based DNA computer of claim 4 wherein: the input
part of mentioned DNA molecular storage corresponds to the blank DNA
molecule and DNA storage unit molecule that contains a known sequence,
while the output part corresponds to a DNA storage molecule having
undergone "superposition operations" obtained through biochemical
processes of enzyme cleavage, enzyme ligation etc.

6. The microfluidic chip-based DNA computer of claim 1 wherein: mentioned
detector performs detection aiming at the DNA output molecule of the DNA
molecular computation unit and mentioned electronic computer issues
commands to the DNA molecular computation unit and DNA molecular storage
with identification and judgment based on the detection results, making
DNA molecule complete the DNA molecular operation and DNA molecular
storage on the operation platform of the microfluidic chips of
computation unit and storage unit respectively.

7. The microfluidic chip-based DNA computer of claim 6 wherein mentioned
detector can be a laser induced fluorescence detector, an electrochemical
detector or an ultraviolet detector.

8. A microfluidic chip-based DNA molecular computation unit of mentioned
microfluidic chip-based DNA computer of claim 1 comprising operation
media, reaction media and the microfluidic chip: mentioned operation
media is the DNA computation molecule with specific sequence, the DNA
transfer molecule with specific sequence used in middle operation and the
DNA output molecule that represents computation results through
biochemical reactions; mentioned reaction media is the various kinds of
biochemical enzymes used in enzyme cleavage and enzyme ligation
reactions; mentioned microfluidic chip has at least enzyme cleavage
reaction area, enzyme ligation reaction area, and result output area
connected by microchannels in sequence, and carries out the liquid
control through the micropump and microvalve.

9. The Microfluidic chip-based DNA computer of claim 8 wherein on
mentioned microfluidic chip, the amount of enzyme ligation reaction
sections correspond to that of the types of transfer molecules in the DNA
molecular computation unit.

10. The DNA molecular computation unit with a microfluidic chip of claim
8 or 9 wherein on mentioned microfluidic chip, a PCR amplification region
is disposed before the result output region.

11. The DNA molecular computation unit with a microfluidic chip of claim
8 or 9 wherein on mentioned microfluidic chip, there are sections for
storing all kinds of operation media and reaction media and these
sections are connected to each correlative enzyme cleavage reaction area
or enzyme ligation reaction area through microchannels.

12. The DNA molecular computation unit with a microfluidic chip of claim
11 wherein on mentioned microfluidic chip, there are sections for storing
all kinds of operation media and reaction media and these sections are
connected to each correlative enzyme cleavage reaction area or enzyme
ligation reaction area through microchannels.

13. The DNA molecular computation unit with a microfluidic chip of claim
8 or 9 wherein on mentioned microfluidic chip, there are sections regions
for storing blank buffer solution and waste solution respectively in a
unified manner.

14. The DNA molecular computation unit with a microfluidic chip of claim
10 wherein on mentioned microfluidic chip, there are sections areas for
storing blank buffer solution and waste solution respectively in a
unified manner.

15. The DNA molecular computation unit with a microfluidic chip of claim
11 wherein on mentioned microfluidic chip, there are sections regions for
storing blank buffer solution and waste solution respectively in a
unified manner.

16. The DNA molecular computation unit with a microfluidic chip of claim
12 wherein on mentioned microfluidic chip, there are sections regions for
storing blank buffer solution and waste solution respectively in a
unified manner.

17. A microfluidic chip DNA molecular storage of mentioned microfluidic
chip-based DNA computer of claim 1 comprising storage media, reaction
media and the microfluidic chip: mentioned storage media includes the
short-chain DNA storage unit molecule with a known sequence, the DNA
blank molecule used in initial operation and the DNA storage molecule
that represents superposition results through biochemical reactions;
mentioned reaction media is the various kinds of biochemical enzymes used
in enzyme cleavage and enzyme ligation reactions; mentioned microfluidic
chip has at least storage unit area, enzyme cleavage reaction area,
enzyme ligation reaction area, and result output area, and carries out
the liquid control through the micropump and microvalve with enzyme
cleavage reaction area, enzyme ligation reaction area, and result output
area connected by microchannels in sequence and storage unit area
connected to enzyme ligation reaction area through microchannels.

18. The DNA molecular storage with a microfluidic chip of claim 17
wherein on mentioned microfluidic chip, a PCR amplification region is
disposed before the result output section.

19. The DNA molecular storage with a microfluidic chip of claim 17 or 18
wherein on mentioned microfluidic chip, there are sections for storing
all kinds of storage media and reaction media, and these sections are
connected to each correlative enzyme cleavage reaction area or enzyme
ligation reaction area through microchannels

20. The DNA molecular storage with a microfluidic chip of claim 17 or 18
wherein on mentioned microfluidic chip, there are sections regions for
storing blank buffer solution and waste solution respectively in a
unified manner.

21. The DNA molecular storage with a microfluidic chip of claim 19
wherein on mentioned microfluidic chip, there are sections regions for
storing blank buffer solution and waste solution respectively in a
unified manner.

Description:

BACKGROUND OF THE INVENTION

[0001] 1. Field of Invention

[0002] This invention provides a Deoxyribonucleotide acids (DNA) computer
with a microfluidic chip technology which carries out enzymatic reactions
to cleave, ligate, and amplify DNA molecule on a microfuidic chip. Using
the DNA molecule as an operation media, the genetic code before one
reaction is taken as the input data, while the genetic code after the
reaction is denoted as the operation results. A novel high speed DNA
computer which possesses tremendous capacity is developed by performing
various controllable DNA biochemical reactions followed by combining and
integrating various chips in the DNA computer.

[0003] 2. Description of the Related Art

[0004] DNA computer is an emerging field that basically combines molecular
biological studies of DNA molecules and computational studies on how to
employ these specific molecules to calculate. The main features of DNA
computer are characterized by its high parallel computing ability, fast
operational speed and enormous data storage capacity. However, the
research on DNA computer until now has encountered the following two
limitations. The first limitation is the lack of fully integrated
hardware device that can support the biological operation-based
computing, confirm the corresponding result and control the correlative
parameters. The second limitation is that these molecular computing
processes are carried out without registering or storing each computation
processes. However, the storage function is one of the main features of
modern computers which are also the essential character that differ a DNA
computer from a DNA computing machine.

[0005] The microfluidic chip technology fully or basically integrates the
fundamental operation units onto a chip with a size of about several
square centimeters where the biological and chemical reactions such as
sample preparation, enzymatic or chemical reactions, product separation
and detection, etc. carry out. The availability of various operation
units and the flexibility to combine them warrant the advantage of
generating chips that can be integrated in large scale. Chip
technologies, in principle, perform the reactions and the separation and
detection of various types of molecules from nucleic acids and proteins
to organic and small inorganic compounds.

[0006] In general, the chip technology includes two major categories. One
is the array micro-porous board chip without circulating network and
separation. This is usually called "biochip", since it is relatively
specific to DNA and protein. The other is based on microfluidic
technology, with a network of microchannels on the chip and controllable
liquid that runs through the whole system. This is usually called "Lab on
a chip", and is the mainstream of chip technology.

[0007] The development of microfluidic chip technology with high
throughputs, integration and strong controllability provides a possible
platform for substituting test tube or surface operation.

BRIEF SUMMARY OF THE INVENTION

[0008] The objective of the present invention is to provide a DNA computer
which uses the microfluidic chip as the operation platform. The invented
DNA computer uses DNA molecules as operation or/and storage media while
employing microfluidic chip as the operation platform of the DNA
molecular computation or/and storage unit. An electronic computer and a
detector are also supplied as the core of a controller.

[0009] The said microfluidic chip includes a DNA molecule computation area
and a DNA molecule storage area. The microfluidic chip comprises the
operation units for restriction enzyme-mediated DNA cleavage,
ligase-mediated DNA ligation, polymerase chain reaction (PCR) and chip
electrophoresis. These operations units are connected by microchannels in
sequence with liquid control running through the micropumps and
microvalves. The controller is connected to the electrodes of the
microfluidic chips of the DNA molecule computation unit and the DNA
molecule storage unit.

[0010] A unique aspect of this invention is the design of specific DNA
sequences to be used in the computation or/and the transfer molecules as
the operation media in a DNA molecule computation area. With such design,
the output DNA molecule can represent computation results after the
biochemical reactions were carried out by various enzymes. The
biochemical reactions used in this invention include restriction
enzyme-mediated DNA cleavage, ligase-mediated DNA ligation and PCR.
Guided by the instruction of the said controller, various kinds of said
biochemical enzymes were chosen and operated to complete the reactions on
the microfuidic chip. The input part of said DNA molecule computation
unit corresponds to the DNA computation molecule and/or DNA transfer
molecule with specific sequence, while the output part corresponds to a
DNA output molecule that represents computation results obtained through
biochemical processes such as DNA cleavage and DNA ligation. A PCR
amplification region is placed in front of the result output region in
order to amplify the signal.

[0011] The DNA molecule storage area in this invention comprises of
storage media, reaction media and the microfluidic chip. The said storage
media includes a short-chain DNA molecule with a known sequence as a DNA
blank molecule in an initial operation, and the DNA storage molecule that
represents superposition results through biochemical reactions. The
reaction media includes various kinds of biochemical enzymes used in DNA
cleavage, DNA ligation and PCR. The microfluidic chip comprises of
operation units of DNA cleavage, DNA ligation, PCR and chip
electrophoresis, which are connected by microchannels in sequence with
the liquid control running through the micropump and microvalve. Guided
by instructions from the said controller, various said biochemical
enzymatic reactions were carried out. The operation processes and the
results were stored in said DNA molecule. The input part of said DNA
storage molecule corresponds to the DNA blank and/or DNA storage molecule
that contains a known sequence, while the output part corresponds to a
DNA storage molecule after performing "superposition operations" obtained
through biochemical processes of DNA cleavage, DNA ligation and so on.

[0012] The said detector in this invention performs detection of the DNA
output molecule on the DNA molecule computation unit. Based on detected
results, the said electronic computer sends commands to the DNA molecule
computation unit and DNA molecule storage unit. These commands further
enable the DNA molecule operation unit and DNA molecule storage unit to
complete the whole reaction processes. Said detector can be a laser
induced fluorescence detector, an electrochemical detector or an
ultraviolet detector.

[0013] Sections for storing all kinds of operation media and reaction
media are designed on said microfluidic chip. These sections are
connected to each corresponding enzymatic reaction region through
microchannels. Regions for storing buffer solution and waste solution,
respectively, in a unified manner on said microfluidic chip are also
provided

[0014] The inventors of this invention use the existing facility to design
and set up a DNA computer with a microfluidic chip, which comprises of a
microfluidic chip, a microfluidic chip workstation and a kit for
completing all kinds of enzymatic reactions. The microfluidic chip is
obtained by the superposition of a flat A with groups of microchannels
and various operation units integrated on one side and a sealed flat B.
Flat A possesses groups of various microchannels and operation units. The
width of the microchannel in the chip is 75 μm. The cross section of
the microchannels is an inverse trapezoid or a rectangle. Channels are
sealed between the two flats, with the inlet and outlet of the channels
set up on flat A. Flat B is a cover plate.

[0015] The said microfluidic chip can be made of glass, quartz or plastic,
wherein plastic chip includes PDMS chip, PMMA chip and PC chip.

[0016] The microfluidic workstation is a set of existing and common work
systems for the microfluidic chip, which consists of the integrated chip
electrophoresis platform, laser induced fluorescence detector, CCD
detector, power supply and computer operating system. It serves the
functions of power supply for the chip, signal collection, and hardware
control of the DNA computer.

[0017] A series of biochemical reagents are needed, in order to make said
DNA computer carrying out functions of input, output, computation and
storage. A kit including a piece of microfluidic chip for DNA computer; a
set of each of restriction endonuclease reagents, ligase reagents, and
PCR reagents; a bottle of electrophoresis buffer solution; and a set of
standard DNA fragments are also included in this invention.

[0018] The restriction endonuclease reagents in this invention include
restriction endonuclease and the reaction buffer solution. The
restriction endonuclease belongs to the class of Fok I, Bgl I, BstX I,
Sfi I and so on. The ligase reagents contain T4 DNA ligase and the
reaction buffer solution. PCR reagents comprise Taq DNA polymerase, the
reaction buffer solution and deoxyribonucleotide triphosphate (dNTP). A
DNA marker with known length is used as the internal standard to
determine the length of the DNA products.

[0019] Over all, this invention unprecedented adopts microfluidic chip
technology to substitute currently used test tube or surface operation in
the DNA computation process. The microfluidic chip technology described
in this invention performs exact and controllable operations, and can be
scaled up to integrate high flux. The invention provides a realistic and
possible platform for constructing a DNA computer within the rigorous
sense.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] FIG. 1 is an illustration of system structure of the DNA computer
based on microfluidic chip.

[0021] FIG. 2 shows a photograph of the microfluidic chip workstation of a
DNA computer.

[0022] FIG. 3 is a schematic view of a microfluidic chip for a DNA
computer. A. a surface plate integrating groups of various microchannels
and the inlet/outlet. B. a cover plate.

[0030] This invention presents a microfluidic chip-based DNA computer
which comprises a microfluidic chip workstation, a microfluidic chip and
a set of kits to hold all kinds of reagents for the enzymatic reactions
(FIG. 1). The microfluidic chip workstation comprises of an electrical
power, a control device and an output device, for the functions of power
supply, signal collection, and concurrently administers the control of
the DNA computer. The high voltage, direct electrical current of the
workstation is connected to eight electrodes where different voltages, if
needed, can be applied to different positions of the microfluidic chip to
control the flow of the reaction solution among the channels according to
demand. The detector of the microfluidic chip workstation can move
correspondently to the chip to detect the reaction products in arithmetic
logic unitblock and storage blockunit, respectively. The chip is the core
of the whole computer. Both the operational computation function and the
storage function of the computer are present on the chip. The DNA
molecules and all kinds of reagents in the reagent boxkit enter the
system through input blockunit.

[0031] An existing device that shows the microfluidic chip workstation of
integrated DNA computer was depicted in FIG. 2. It possesses two driving
modes of electroosmosis and pressure, uses laser induced fluorescence as
the detection means, and includes the laser induced fluorescence
detection, CCD image supervision optical system, eight-electrode direct
current high voltage power source and software system. The upper side of
the microfluidic chip workstation is the chip's fixed platform and
electrode operation platform which can move up and down. The down side
comprises of integrative optical detection system, including CCD for
focusing and channel supervision as well as the optical detection system.
The region of narrowband filter is designed to be replaceable for
multiple wavelength choices. The rearward of the workstation is comprised
of switchable high voltage power source and related circuits. The hard
core of DNA computer where groups of complicated microchannels are
integrated--the microfluidic chip--was shown in FIG. 3. It prosecutes
functions of input, output, computation and storage; integrates the
operation units of DNA cleavage and ligation reaction, PCR and
electrophoresis separation. The reservoirs and microchannels of Group (a)
prosecute the input, output and the computational function of DNA
computer while the reservoirs and microchannels of Group (b) complete the
function for storage.

[0032] The computationarithmetic logical unit (a) is on left hand side of
the chip, as shown in FIG. 3 (a) and detailed in FIG. 4. Reservoir (1) in
FIG. 4 is the restriction endonuclease DNA cleavage reaction chamber. It
also serves as the input unit of DNA computer for receiving all
instructions. The restriction endonuclease DNA cleavage reaction chamber
(1) is connected to two DNA ligation reaction chambers (2) and then two
PCR chambers (3) in sequence. The buffer reservoir (4), the waste
reservoir (6) and two standard DNA fragments reservoir (5) comprises a
detection region of a cruciform channel. The injection channel is between
the two standard DNA fragments reservoirs (5), and the detection channel
is between the buffer reservoir (4) and the waste reservoir (6). (7) And
(8) represent a microvalve and a micropump, respectively, which control
the connectivity between each operation unit. PCR chambers (3) are
connected to the injection channel of the detection region. The detection
point is the fan-outoutput point. It detects the DNA molecule by laser
induced fluorescence, transmits signals to the software of the
workstation through ND transformation and then translates and expresses
for output. The channels and reservoirs in the chip are necessary
functional unit for finishing DNA computing, realizing biochemical
reactions of the DNA, separating and detecting the reaction products in
time, ensuring the input, output and completing the computational
function of DNA computer.

[0033] On the storage side of the chip, as shown in FIG. 3 (b) and FIG. 5,
a "stack" storage is designed to store the results each computation step
generate. This "stack" storage plays a relatively important role in
identifying independent context. As shown in FIG. 5, the DNA storage unit
comprises two storage unit molecule reservoirs (9), a DNA cleavage and
DNA ligation reaction chamber (10), a PCR chamber (3), a buffer reservoir
(4), a waste reservoir (6), a sample waste reservoir (12). The DNA
cleavage and DNA ligation reaction chamber (10) is connected to two
storage unit molecule reservoirs (9) and the PCR reaction chamber (3),
respectively. The PCR chamber (3), the sample waste reservoir (12), the
buffer reservoir (4) and the waste reservoir (6) form a detection region
of a cruciform channel, while the detection channel is between the buffer
reservoir (4) and the waste reservoir (6). The injection channel is
between the PCR chamber (3) and the sample waste reservoir (12). (7) And
(8) are the micro-valve and the micro-pump, respectively, which control
the connectivity between each operation unit.

[0034] As shown in FIG. 6, the kits used in the microfluidic chip-based
DNA computer_include a piece of microfluidic chip for DNA computer (11),
a set of restriction endonuclease reagents (22), a set of ligase reagents
(33), a set of PCR reagents (44), a bottle of electrophoresis buffer
solution (55) and a set of standard DNA fragments (66). The restriction
endonuclease reagents include restriction endonuclease and reaction
buffer solution. The restriction endonuclease belongs to the class of
FokI, BglI, BstXI, SfiI and so on. Ligase reagents contain T4 DNA ligase
and reaction buffer solution. PCR reagents comprise Taq DNA polymerase,
reaction buffer solution and deoxyribonucleotide triphosphate (dNTP). DNA
marker with known length is used as the internal standard substance to
determine the length of the DNA products.

[0035] This invention is unique as functions of each composition unit of
the microfluidic chip-based DNA computer (shown in FIG. 1) are compared
with those of the typical electronic computer. There differences are
summarized in Table 1.

TABLE-US-00001
TABLE 1
the comparison between functions of each component in DNA computer and
those of
the typical electronic computer
Input Output Operation Memory Control
Unit Data input Data display Data processing Data storing in coherence and
the process of harmony
operation in all part
Electronic Input equipment: Output equipment: ALU EMS memory CPU
computer Keyboard, Mouse CRT
MC DNA Specific DNA A collection of Various kinds Memory chips Control
Computer molecule and illustrative plates of response and the DNA
procedure
corresponding of the response systems DNA molecule designed
reagent in the react molecule detected computing storing the according to
the
chamberpool by the work station required information process of DNA
computer

[0036] The functions of the microfluidic chip-based DNA computer are
described below:

[0037] For convenience, the finite state automaton with two input symbols
of a, b and three states of S0, S1, S2 is adopted to
illustrate functions of the microfluidic chip-based DNA computer in FIG.
7. The finite state automaton is based on syntax pattern recognition of
isosceles triangle.

[0038] In general, a triangle can be regarded as being composed of several
line fragments of the same length, as shown in FIG. 8. Such line
fragments are the fundamental components of a triangle, which includes
horizontal line, ascending oblique line and descending oblique line.
Therefore, a triangle represents character strings made up of units. For
instance, the triangle illustrated in FIG. 8 can be expressed as
"aabbbcccc". In this invention, final state was obtained by DNA
computation based on the above-mentioned finite state automaton.
Comparison was then made between the two sides of the object. If the
final state is S0, both sides of the triangle are equal, and vise
versa.

[0039] The formula for corresponding state transfer of the finite state
automaton in FIG. 7 is designed as:

##STR00001##

[0040] The transfer molecules are designed as:

##STR00002##

[0041] The twenty (20) base pairs on the left of the transfer molecules
are assembled into different sequences.

[0042] The blue print of the finite state automaton of the microfluidic
chip for a DNA computer is depicted in FIG. 3, which illustrated the
principle and the processes of functions of input, output, computation,
control and storage.

[0043] For example, when DNA molecules and the corresponding reaction
reagents are added into the enzymatic cleavage reaction reservoir (1) in
FIG. 4 as input data: [0044] {circle around (1)}. The DNA molecule
completes DNA cleavage, DNA ligation, and PCR in reservoirs (1)-(3)
respectively, and carries out the DNA computing. [0045] {circle around
(2)}. Electrophoresis separation is carried out in the channels between
reservoir (4) and reservoir (6). Then the data is exported. [0046]
{circle around (3)}. According to the exported data, the storage unit
shown in FIG. 5 performs data storage. [0047] {circle around (4)}.
Various storage unit molecules are put into reservoir (9) in FIG. 5. DNA
cleavage and ligation reaction, as well as PCR are completed in reservoir
(10) and reservoir (3), respectively, to perform storage. [0048] {circle
around (5)}. The reaction products is separated and detected in the
channels between reservoir (4) and reservoir (6). The storage results
were recorded.

[0049] A detailed description of how to perform the five major functions
of DNA computer in the finite state automaton on the microfluidic chip is
shown below:

(1) Input

[0050] Symbols of the input molecules in the finite state automaton in
FIG. 7 are as follow:

TABLE-US-00002
a: ATCACG b: ACGGTA
TAGTGC TGCCAT

[0051] Terminator Molecule:

TABLE-US-00003
GTACCT
CATGGA

[0052] For example, if the finite state automaton with the initial state
of "S0" and input symbol of "aabbb", the corresponding DNA input
molecule is obtained as the following:

##STR00003##

[0053] The solution containing the above-mentioned DNA sequence is guided
into the reservoir (1) of the chip on the computing computation unit
(Group (a) side a in FIG. 3) and the input process is performed.

(2) Output

[0054] The restriction endonuclease of FokI is chosen. Its recognition
site is 5' . . . GGATG(N)9 . . . 3' and the enzymatic cleavage site
is located at the end of 9th nucleotide. After enzymatic cleavage, a
4 bp sticky end (the 9th to 13th nucleotide on the 5' end of
the opposite strand) is formed with its sequences vary according to the
combination of different states and symbols. Table 2 depicts such a
combination.

[0055] Each sticky end formed by enzymatic cleavage is ligated to a
transition molecule with a complementary sticky end of the enzymatic
reaction carried out by T4 DNA ligase.

[0056] The output-detecting molecule is designed to detect the
corresponding states resulted from detecting program. Thus, the finite
state automaton of each terminator state is designated to a corresponding
output-detecting molecule as follows:

##STR00007##

[0057] The output-detecting molecules and output-molecules joined together
to form a report molecule which is detected and recorded in reservoirs
(4)˜(6) on the chip, as shown in FIG. 4.

(3) Computation

[0058] The calculating computational procedure and corresponding
electropherograms of finite state automaton with an "aabbb" input symbol
was shown in FIG. 9. In FIG. 9, (a)-(g) designate the corresponding
electropherograms of input state, each intermediate state and output
state. FIG. 9 (h) On the right hand side is the DNA sequential diagrams
to show the computing procedure for the "aabbb" input symbol. FIG. 9 (a)
is the electropherogram of the input molecule "aabbb". FIG. 9 (b)-(f)
show electropherograms of the intermediate resulting molecules generated
during the computational process. FIG. 9 (g) is the electropherogram of
output molecule. We identify the length of every specific DNA molecule
(labeled as Input-aabbb, T1-aabbb, T2-aabbb, etc.) with 100 bp series
marker as the internal marker. The peaks of the 100 bp ladder (with
increasing migration time) represent the DNA molecules with 100, 200,
300, 400, 500, 600, 700, 800, 900, 1000, 1500 bp in length, respectively.
The 500 bp peak in the ladder is significantly higher than others, thus
it is used as a marker in the electropherograms. The peak of all specific
molecules of FIG. 3 9 (a)-(g) had obviously changed their positions
relatively to the peaks of marker, indicating the length of the DNA
molecule has changed after enzymatic digestion and ligation.

(4) Storage

[0059] The storage is completed by the data structure of "stack" in the
microfluidic chip-based DNA computer. According to the computational
results shown by the transfer molecule and the corresponding symbol, the
microfluidic chip workstation controls the storage chip to record the
corresponding data into memory molecules, and executes the storage
function.

(5) Control

[0060] The results of computation are fed back to the workstation. A
pre-designed computer program controls the storage unit on the right hand
side of the chip which stores the corresponding data in memory molecules.
The sequences of the events are depicted as follows: [0061] the signal
that reflects the information of DNA molecule is sampled into the MC
workstation via an A/D converter; [0062] the sampled signal data are
converted into graphic file; [0063] the migration time of a DNA molecule
required to migrate is proportional to its length. Therefore, the length
of the PCR product can be calculated by analyzing the electropherogram of
the PCR product mixing with DNA markers in a computer program. The
transition molecules in each reaction are identified by the position of
the peaks; [0064] the result is displayed and a signal is sent to the
data-storing endstorage unit of the DNA computer. The data information of
each reaction step is stored via DNA molecules; [0065] the computing
computation unit of microfluidic chip on FIG. 1, side Group (a) transmit
the signals to the workstation which subsequently connect and control the
storage unit on side Group (b) (FIG. 1) until all orders are sent.

[0066] One of the biggest challenges in the field of DNA computing up to
now is to build storing storage units that are capable of storing all the
intermediate states and results of the calculation. This obstacle is
unlikely to be resolved by conventional in-test-tube protocols. On other
hand, our microfluidic chip technology is capable of solving this
problem.

[0067] In detail, data structures like table, stack and queue are of vital
importance to DNA computers as well as to conventional computers.
Inputting symbol of "aab" is used as an example to illustrate how to
generate the stack storing on microfluidic chip. We assume that the
initial state of the storage unit is "empty (E)" (see FIG. 10), in other
words, the bottom of the stack is filled with "E". As mentioned before,
Memory-a or Memory-b adds a 13 by or 21 by sequence, respectively, to the
blank molecule "E". The results of the storage could be obtained via the
length or sequence detected at the end.

[0068] Molecule E was designed by amplifying the plasmid PUC 19 (TaKaRa
Biotechnology Co., Ltd) with primer L1 and R1 which results in a DNA
fragment of 304 bp. This DNA fragment, at the location of 417-422 (based
on PUC 19 sequences), contains the recognition site of restriction
endonuclease BamHI: GGATCC. A sticky end on its left hand side of the E
molecule is obtained by BamHI cleavage of the DNA molecule.

##STR00008##

[0069] To design the storage unit molecule in the stack, the sticky end on
the right hand side of Memory-a and Memory-b molecules will be annealed
with the sticky end of the blank molecule "E" after BamHI cleavage first.
The intermediate products will then be cleaved by FokI again because they
comprise the recognition site of FokI. Storage is based on the state and
the symbol of the transition molecule encoded. As mentioned before,
Memory-a or Memory-b adds a 13 by or 21 bp, respectively, to the blank
molecule "E" until the output of the terminator molecule. The results of
the storage could be obtained via the length or sequence detected at the
end.

[0070] A detailed operation process of storage is described as follows:

[0071] Firstly, the E molecule with a sticky end on its left hand side is
obtained by the restriction endonuclease BamHI cleavage of DNA molecule
at 30° C. in chamber (10) (FIG. 5). The length of the E molecule
is detected to be 263 bp. The temperature is then raised to 65° C.
for 10 min to deactivate BamHI. Memory-a or Memory-b is introduced into
chamber (9) (FIG. 5) according to the result of calculating, which will
then ligate with E molecule at 18° C. for 30 min. The ligase is
then deactivated at 65° C. for 10 min. The DNA product after
ligation is used as a template for PCR amplification, and the product is
detected by electrophoresis. The above-mentioned processes are repeated
until the output of terminator molecule, which signals the completion of
storage. The final storage molecule encodes the information of DNA
computing, which can be read out at any time.

[0072] Using "aab" input symbol as an example, the dynamic storage process
of the finite state automaton is described as follows. The schematic view
of the stack storage process and the corresponding electropherograms of
the product of each storage step are depicted in FIG. 10 (on the left
hand sidea). The corresponding computational process was shown on FIG. 10
on the right hand side(b). The transfer molecule contains the information
of states and symbols during the computational processes. The transition
molecules employed in the finite state automaton with the input symbol of
"aab" are T1, T2, and T3. Their corresponding symbols are "a", "a" and
"b". Memory-a and Memory-b molecules are joining to E molecule
sequentially, forming Ea, Eaa and Eaab and achieving stack storage of
input "aab". Methods similar to stack storing process can also be
utilized to achieve dynamic storing processes of table and queue.